Industrial Research and Development on the Production Process and Quality of Cultured Meat Hold Significant Value: A Review

Donor selection is the most fundamental aspect of the production process, involving considerations such as the breed, sex, and age of the animal and the specific body part from which the cells are sourced (Stephens et al., 2018). As shown in Table 1, cultured meat is being produced from cells sourced from various types of animals. Currently, a significant number of commercialized products derived from this process have been developed and are available to consumers (Lee et al., 2022a). For these products, the cells are primarily sourced (in descending order of usage) from cattle (25%), poultry (22%), seafood (19%), pigs (19%), and other animals (15%; Choudhury et al., 2020). Cattle and poultry are predominantly used for research purposes and most of those researches are targeted at religious consumers (Bryant, 2020).

Also, many consumers and scientists commonly know that cultured meat has high advantages for religious reasons and the standard of cell selection is influenced by its reasons. However, for example in the Islamic community, the main point of choosing meat is “Does the meat (cultured meat) produced follow the halal status?” and this point shows that cultured meat isn’t always suitable for religious people (Chriki and Hocquette, 2020). Furthermore, Siddiqui et al. (2022b) reported that socially conservative consumers expressed negative reactions towards cultured meat, and some religious communities, such as Hindus, expressed vegetarianism is regarded as superior to meat eating. These discussions bring the new research development of cell selection and collection techniques from animal bodies and many new studies have to be started.

Once the livestock breed is selected, the next step involves selecting factors such as sex, age, and specific parts of the animal. This decision is dictated by the quality of the satellite cells in the collected muscle tissue (Skrivergaard et al., 2023), which is determined by assessing factors such as their yield and differentiation capacity (Arshad et al., 2017). This assessment is conducted to select the most suitable tissue for meat cultivation. Determining the quality of satellite cells is crucial because the cells play a pivotal role in the regeneration of the muscle tissue that has been damaged through injury (Hong et al., 2021), making them the most critical factor in the cell selection process. Kim et al. (2023c) reported that many factors affect cultured meat production and there are existing unfigured mechanisms that need research. Coles et al. (2015) reported that the breed of origin, live weight at slaughter, and carcass weight affect the collected cell proliferation and this seems that differential gene expression is the main reason for these phenomena.

For these reasons, the final product of cultured meat is affected by the cell donor animal’s genetic characteristics, some researchers are proposing to establish optimized cell models in genetic engineering tools concerning genetically modified organisms (GMOs) (Martins et al., 2023). Also, some researchers found out that cultured meat is more suitable for their Swiss sample compared to GMOs food and this could be a key point for getting balance in the genetic engineering side of cultured meat (Bryant and Barnett, 2020). This describes that many new studies can be excavated in the donor selection part and could be additional scientific data for the traditional meat industry.

Cell separation is the process by which the satellite cells are efficiently isolated from the muscle tissue (which comprises various cell types, including muscle fibers and stem cells; Li et al., 2022b). This process ensures that only satellite cells are obtained from the tissue. Typically, after the initial separation through physical and chemical dissociation, secondary separation is performed using methods such as filtration and centrifugation, density gradient centrifugation, and cell separation based on the antigen–antibody reactions of surface markers (Swatler et al., 2020). Two commonly used cell separation methods are fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) (Table 2).

FACS utilizes antigen–antibody reactions to recognize surface markers on cells as antigens, which have been pre-labeled with fluorescent substances to facilitate the cell sorting process. A flow cytometer is used to separate the cells, allowing for the precise analysis of their size and internal structure (Kim et al., 2022b). Furthermore, the integration of FACS with sequencing, known as FACSeq, proves to be highly effective. This approach enables the detailed exploration of individual cell physiology, facilitating the identification based on factors such as relative nucleic acid contents and cell membrane integrity (Dridi et al., 2023). Recently, owing to the meticulous nature of the FACS method, certain researchers have devised a FACS strategy specifically for purifying adipose progenitor cell (APC). Subsequently, they demonstrated that the purified APC exhibited a notable capacity for proliferation and adipogenic differentiation (Song et al., 2022).

Similarly, MACS relies on antigen–antibody reactions, but antibodies with magnetic properties are used instead to react with antigens on the cell surface. Cells with attached antibodies are then separated using a magnet. This method facilitates rapid cell separation and high cell viability (Choi et al., 2020a). Hence, MACS is considered less disruptive in the separation process compared to FACS, making it a more suitable choice for large-scale expansion (Kim et al., 2023a). While FACS incurs significant costs for both entry and maintenance and exhibits slow speed, hindering high-throughput sample handling, bead-based MACS is a solution to these issues. Nonetheless, magnetic-based approaches grapple with challenges such as low specificity (stemming from the use of a single antibody type) and difficulties in scaling up samples due to the intricate relationship between magnetic field strength and distance (McNaughton et al., 2022).

Taking advantage of the strengths of both FACS and MACS, a hybrid approach that combines these two techniques for cell separation is being widely used in research pertaining to cultured meat production (Guan et al., 2022). In combining two techniques, the strengths of FACS, known for its multiple labeling and sorting capabilities, and MACS, appreciated for high throughput and quick sorting times. Kang et al. (2021) reported they developed an immunomagnetic microfluidic integrated system (IM-MIS) that achieves high yield, high throughput, and minimal loss based on the differentiated cell phenotype.

With the ongoing advancements in these technologies, there is an anticipation that cell separation technology will stabilize, facilitating swift industrial progress in the field of cell sorting.

Cell culturing primarily involves the use of proliferation methods to increase the number of selected cells (Fig. 1). Various substances, such as basal culture medium, serum, growth factors, and antibiotics, are used to provide the necessary conditions for cell regeneration and maturation during this process (Siddiqui et al., 2022b). Basic culture media, such as Dulbecco’s modified Eagle’s medium (DMEM), contain essential nutrients to support and maintain the growth and health of the cells while exponentially increasing their numbers. DMEM offers several advantages, such as commercial availability and a bio-mimicking environment enriched with ingredients like amino acids and vitamins. Consequently, DMEM addresses challenges associated with time-consuming preparation, as well as various issues related to precipitation and storage (Bayrak et al., 2020).

Fig. 1. The whole process for manufacturing cultured meat. MACS, magnetic-activated cell sorting; FACS, fluorescence-activated cell sorting.

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Any deficiencies in the basic medium are supplemented with additives, such as a specific serum, growth factors, and antibiotics (Zhang et al., 2020). Specifically, animal-derived sera, such as fetal bovine serum (FBS), are crucial for cell cultures because they are highly effective in promoting cell proliferation (Post et al., 2020). FBS, naturally tailored for the prenatal development of unborn calves, boasts an extensive array of nutrients, growth factors, and adhesion factors with minimal antibody content (van der Valk, 2022). Its historical preference stems from its relatively low cost and widespread availability, making FBS the primary choice for supplementing nearly all eukaryotic cell culture media. However, demand for alternatives to sera is growing, owing to the ethical concerns and high costs associated with their use. In recent years, various blood-free additives, such as B-27^TM and Xerum Free^TM, have been developed to replace FBS (Xin et al., 2021). These products aim to minimize animal sacrifice and reduce the cost of cultured meat production. Furthermore, to alleviate concerns regarding the consumption of antibiotics and anti-inflammatory agents in the final cultured meat products, some producers have opted for methods that do not use these unwanted bioactive molecules. However, this approach requires delicate culture control, as it can lead to a sharp decrease in cell viability (Piochi et al., 2022).

Microcarriers, an optional material for cell culturing, are formed into beads and have been established as an expanded growth surface to support the differentiation and proliferation of various types of cultured cells (Norris et al., 2022). And there are edible, non-edible, and degradable microcarriers exist, among those kinds, edible microcarrier is most preferred and it is classified into polysaccharides, lipids, polypeptides, and composites/synthetics (Bodiou et al., 2020). The importance of edible microcarriers is to reduce the final cost of cultured meat products by increasing cell harvest yield (Zernov et al., 2022).

The most critical environmental factor in cell culturing is temperature, as it is essential for cell culturing. Mass cell culturing is predominantly carried out in bioreactors, where optimal cell culture is conducted at a temperature of 37°C, mimicking the human body, and supplied with oxygen (Garrison et al., 2022). Guan et al. (2022) reported that mildly elevated temperatures (39°C) and mechanical stimulation are among the environmental cues that have been proven to boost both myogenic differentiation and hypertrophy. Some environmental cues like mild high temperature (39°C) and mechanical movement have also been demonstrated to enhance myogenic differentiation and hypertrophy (Guan et al., 2022). Consequently, while inducing heat stress through elevated culture temperatures may not independently suffice for cell growth and differentiation, it can effectively promote growth factor-mediated cell proliferation and differentiation (Oh et al., 2023).

Taking these aspects into consideration, both in cell culture and collection, it becomes imperative to align with the ethical consumption tendencies of consumers. Simultaneously, there is a continuous need to explore avenues that provide industrial economic advantages.

In cell structuring, the main point is to stabilize the differentiation of muscle cells. It is also called subsequent hypertrophy and this is the mix of biochemical and mechanical stimuli (Post, 2012). A scaffold structure is necessary for organizing the cultured cells into tissues. To reproduce all important features of conventional meat, the set of requirements for biomaterials used to produce cultured meat is highly specific (Wollschlaeger et al., 2022). The material should be edible, sustainable, widely available, animal-free, non-toxic, cheap, processable, and ideally have none or only a mild taste.

Animal-derived scaffolds, which are primarily composed of collagen, have the advantage of providing minimal heterogeneity during cell cultivation. Furthermore, they contribute to the texture and flavor of the final product, aiding in replicating the characteristics of conventional meat (Seah et al., 2022). Collagen gels or collagen–Matrigel complexes are commonly used because they enhance protein production (Post, 2012). Collagen stands out as a well-established material for cell adherent coatings in tissue engineering. Considering that HC peptides share the identical amino acid sequence with collagen and retain cell-binding capability even after collagen denaturation into gelatin, it is reasonable to anticipate robust cell adhesion on hydrolyzed collagen surfaces (Koranne et al., 2022).

Plant-based scaffolds, which are existing plant structures onto which the cultured cells can be attached, offer the simplest means to achieving cellular myogenesis. Additionally, they allow for the consumption of nutrients naturally present in plants, providing an added advantage (Levi et al., 2022). Decellularized spinach is a representative plant-based scaffold that shows high cell adhesion and survival rate and forms suitable cost on the industrial side (Jones et al., 2021). To reproduce the structure of muscle tissues in decellularized spinach scaffolds, the critical factors include the precise composition of the tissue, the arrangement of cells within the scaffold, and the influence of surface topography and cell origin, which may vary based on plant species and leaf position (Rao et al., 2023). However, plant-based scaffolds, which may include polysaccharides such as cellulose, alginate, and hyaluronic acid, carry the risk of inducing allergies (Djisalov et al., 2021), rendering them less suitable for consumption by vulnerable consumers.

Recently, interest in the use of 3D printing technology has been growing, and research studies on the use of 3D printers to produce scaffolds and to directly create cultured meat in the shape of conventional meat are underway (Ramani et al., 2021). In 3D bioprinter, the nozzle size, extrusion pressure, and source of filler highly affect the final products of cultured meat (Djisalov et al., 2021). The main strength of 3D printing technology is the creation of free forms, allowing researchers to realize the desired shape with a high realization rate and freely adjust the type and proportion of the structure (Li et al., 2021). Also enhancing tissue distribution of macromolecules and cells, this technique contributes to producing final products with improved organoleptic properties, offering precise deposition of cells, micronutrients, technological aids, and biomaterials in predefined locations and shapes, presenting advantages over alternative biofabrication methods (Barbosa et al., 2023).

While these aspects greatly aid in the differentiation of cells cultured on the scaffold into muscle, it seems essential to establish cell classification and safety verification methods that align with the scaffold’s characteristics.

Various technological development studies have been conducted aiming to achieve comparable nutritional components, such as protein, essential amino acids, vitamins, and mineral content, in cultured meat compared to conventional meat, from a nutritional perspective (Fraeye et al., 2020). The nutritional quality of cultured meat is influenced by the basic culture medium, serum, growth factors, and other nutrients used in the cell culture. Various studies are underway to investigate the nutritional composition and content of the products (Chriki and Hocquette, 2020). As of now, the protein content (the main reason why people eat meats) of cultured meat has not been quantified; however, morphological observations suggest similarities to traditional meat in terms of cytoskeletal proteins, with current research focusing on optimizing the nutrient content of the growth medium to promote the development of cells with higher protein content (Broucke et al., 2023). So huge differences appear in other nutrient contents except protein contents between traditional meat and cultured meat.

The type and content of fat in cultured cells can be adjusted according to the manufacturer’s preference or purpose, and, like muscle cells, they must undergo a separate differentiation process during cultivation (Fish et al., 2020). Fraeye et al. (2020) reported that the nature of the production process rendered regulation of the fat composition of cultured meats possible, thus allowing for the development of healthier products through adjustments of the essential fatty acid, polyunsaturated fatty acid, and trans-unsaturated fatty acid ratios and calorie content. Accumulating as storage compounds in animal muscles, conventional meat is a nutritionally dense food rich in high-quality proteins, as well as a diverse array of vitamins and minerals (Singh et al., 2022). Meat blood is abundant in various nutrients, particularly minerals like calcium, iron, magnesium, potassium, and sodium (Lee et al., 2022b). Therefore, consuming meat not only provides essential nutrients directly but also includes minerals that are present in the blood.

However, in cultured meat, nutrient contents such as vitamins, minerals, etc. are affected by serum. The composition and quantity of serum used can vary depending on the donor’s biological information, diet, and lifestyle (Lee et al., 2022b). Therefore, even the same type of serum can have differences in components and amount. Kadim et al. (2015) reported that in cultured meat, the essential amino acids, minerals, vitamins, and bioactive compounds provided by the basic culture medium, serum, and other nutrients used during cell culture were similar to or even exceeded those in conventional meat, demonstrating the nutritional advantages of meat cultivation. Currently, Ultroser G serves as a commercially available serum-free growth medium, acting as a substitute for fetal bovine serum. It encompasses all the essential nutrients required for eukaryotic cell growth, including growth factors, binding proteins, adhesion factors, vitamins, hormones, and mineral trace elements (Jairath et al., 2021).

Therefore, cultured meat maintains its nutritional quality and can even contain enhanced contents of nutrients such as essential amino acids and fatty acids that may be lacking in conventional meat. The meat culturing process, thus, allows for the production of products with high nutritional value.

The latest research on textural properties has exposed suboptimal structuring and texture attributes in manufactured cultured meat (Starowicz et al., 2022). Notably, non-instrumental studies profiling texture has centered on sensory characteristics, including hardness, springiness, and chewiness (Yuliarti et al., 2021). Li et al. (2022a) reported that meat cultured on edible 3D chitosan–sodium alginate–collagen/gelatin scaffolds had similar textural characteristics (e.g., chewiness, springiness, and resilience) as those of conventional meat of the same weight, a finding they attributed to the comparable fibrous characteristics of both products. Furthermore, in a study on cultured meat production using pig muscle stem cells, Zhu et al. (2022) found that the addition of l-ascorbic acid 2-phosphate during the cell culture phase led to increased expression of the myosin heavy chain protein and differentiation genes, which resulted in enhancement of the tissue texture. Moreover, in their research on cultured meat using smooth muscle cells, Zheng et al. (2021) observed that the texture of the final product was significantly influenced by the collagen content. They found that the co-culturing of smooth muscle cells with hydrogel and formation of a network structure enhanced the texture of cultured meat. This indicates that, aside from the characteristics of the cultured cells themselves, the type of scaffold and additives used can also affect the texture of the final product. Tomiyama et al. (2020) found that among various scaffold structures, those mimicking the striped texture resembling muscle architecture promote myotube formation.

Also, some scaffolds can undergo breakdown and reconstruction by cells, in general, maintaining the structure and mechanical properties of the scaffold has a significant impact on the texture of cultured meat (Langelaan et al., 2010). In light of this, there is a trend in developing scaffolds using edible materials such as alginate, gelatin, collagen, and starch, taking advantage of the characteristics of the scaffold. Among various scaffolds, animal-derived ones are suggested to more closely mimic the traditional texture of meat compared to plant-based scaffolds (Levi et al., 2022). Paredes et al. (2022) compared the textural properties of commercially available conventional sausages and sausages made from cultured meat and found that the hardness, cohesiveness, springiness, chewiness, and gumminess of the two products were similar. This finding suggests that cultured meat products are similar to conventional meat products in terms of textural quality, highlighting the potential for future expansion into the development of cultured meat-based products. However, in the case of cultured meat with a meat-like structure rather than a processed meat form, currently available products for commercial sale have generally received lower consumer evaluations compared to traditional meat (Kim et al., 2022a).

It is particularly suggested that ongoing efforts are needed for further improvement in texture, especially in terms of consistency.

Intrinsic qualities such as taste, texture, smell, and nutritional value constitute the importance of meat. These essential attributes play a critical role in influencing consumers’ choices when it comes to purchasing and consuming meat (Rombach et al., 2022). Furthermore, sensory properties are more treated as main factors than price, health function, and convenience, and if the sensory properties are not well possessed, consumer rejection rapidly increases (Pakseresht et al., 2022). The lipid oxidation products of conventional meat interact with the products of the Maillard reaction, creating a complex flavor profile that contributes to the meat color and taste (Chen et al., 2022). Therefore, for the flavor of conventional meat to be replicated in cultured meat, an understanding of how well the product can mimic the taste of fats is needed (Ng and Kurisawa, 2021).

Further research on the mechanisms of flavor compounds is necessary. Broucke et al. (2023) reported various studies that are using different methods to enhance the flavor of cultured meat, including co-culturing adipocyte precursors with muscle cells and adding carotenoids during the cell culture phase, with a focus on flavor precursors. Additionally, Louis et al. (2023) investigated the regulation of the fatty acid composition in adipose-derived stem cells from Wagyu cattle and found that the initial lipid composition can be controlled by adjusting the fatty acids during the cell differentiation process when producing fat cells. This resulted in a fat composition similar to that of conventional meat. These studies indicate that a foundation for replicating the flavor of fats in cultured meat has been established and underscore the need for continued in-depth research specifically focusing on fat cells. Joo et al. (2022) conducted a comparative study of cultured and conventional meats using electronic nose analysis. The researchers observed that traditionally produced meat was superior in terms of flavors such as umami. Also, Rolland et al. (2020) reported that a contrast in taste was evident between the conventional and ‘cultured’ hamburgers during the sensory evaluation of six attributes, with the ‘cultured’ hamburger receiving a slightly favorable assessment.

This superiority was attributed to differences in the maturity of muscle fibers, implying that the flavor of the final cultured meat can be influenced, even during the initial cell selection phase of primed cultivation. All the above findings underscore the need for further research on the combinations and ratios of different types of muscle and fat cells. Verbeke et al. (2015) reported that significant challenges lie in advancing both the product and its production process to closely emulate traditional meat, especially concerning sensory characteristics and pricing.

Additionally, challenges involve scaling up the process for enhanced resource efficiency and cost-effectiveness, along with addressing regulatory and intellectual property issues.

Cultured meat is produced in a sterilized environment free of contaminants, making it generally safer than conventionally produced meat, in terms of microbial contamination. However, proper handling, processing, packaging, and storage practices after production need to be maintained (Siddiqui et al., 2022a). Upon introducing cultured meat to the market in the EU, regulations from the Genetically Modified Food and Feed Law have been applied, encompassing areas such as labeling, official control of animal-derived products, and microbiological criteria (Ketelings et al., 2021). Similar to other food production processes, ensuring safety throughout the entire cultured meat production process in the EU requires the implementation of food safety monitoring systems like hazard analysis and critical control points.

Maintaining the storage stability of cultured meat serves not only the purpose of protecting consumers’ health from microorganisms but also aims to prevent changes in the texture characteristics of the final product, which could impact the tissue structure (Rubio et al., 2020). Ong et al. (2023) reported that the microbial composition of the final product is influenced by the indigenous microbial population in the production environment. Therefore, the post-production microbial composition of cultured meat is anticipated to be similar to that of the indigenous microbial population in the production environment. Additionally, in their study on cultured meat with added carotenoids, Stout et al. (2020) found no significant difference in malondialdehyde values between days 0 and 1 before heating of the regular cultured meat samples; however, after heating, approximately two-fold difference was observed in malondialdehyde values between days 0 and 1. This indicates that the storage conditions, form, and method greatly influence the cultured meat after its production.

In particular, an analysis of the factors that lead to significant changes in meat stability after heating is needed, and the implementation of appropriate storage methods is required. Furthermore, Singh et al. (2022) reported that utilizing the fermentation characteristics of organisms such as mushrooms, yeast, and fungi enhances the taste profile of cultured meat and extends its shelf life. This suggests that the use of natural antimicrobials will increase in the future. Considering that cultured meat is primarily generated in a laboratory environment, it can be regarded as less prone to zoonotic diseases than conventional meat products. However, there are knowledge gaps in the current understanding of food safety concerning cultured meat, particularly because the majority of research endeavors are concentrated on optimizing production methods (Hadi and Brightwell, 2021).

Therefore, future research studies should focus on utilizing various additives to enhance the shelf life of cultured meat while simultaneously improving other characteristics, such as flavor, texture, and nutrition.